Our studies in this area have focused on two specific effects of Hcy on immune cells and their function, namely cellular apoptosis and cytokine expression. Hcy has been shown to induce apoptosis in a number of cell lines and cell type including immune cells. We have found that treatment of resting and activated human and murine T cells with L-Hcy results in a dose-dependent increase in apoptotic cell death. The L or D,L forms of Hcy was more potent than Hcy thiolactone in this respect, while S-adenosyl Hcy (SAH) was found to be significantly less active. We also found that the pro-apoptotic effects of Hcy were abrogated with the addition of serum, pan- and specific-caspase inhibitors and PARP inhibitors to the cultures. These results suggest that L-Hcy, like other apoptotic stressors, leads to the activation of the caspase cascade and eventually to the cleavage of the key cellular proteins facilitating the release of mitochondrial cytochrome c, DNA fragmentation, and eventually leading to the typical morphological changes observed in cells undergoing apoptosis. In addition, in vivo infusion of L-Hcy but not SAH induced a dramatic reduction in thymocyte numbers (in particular CD4+CD8+ T cells), thymic weights and a dramatic change in thymic morphology. While these findings are provocative, a specific role for Hcy in age- or disease-associated thymic atrophy remains to be defined. We are currently writing up this work for publication. In addition, we have found that activation of purified human or murine T cells in the presence of L-Hcy or thiolactone but not SAH results in a significant increase in the expression of several Th1 and Th17 but not Th2 cytokines. These effects were also observed upon in vivo infusion of Hcy into normal and TCR transgenic mice. In addition, we have found that immune cells actually produce the various forms of Hcy upon activation and that these various Hcy appear to play an endogenous regulatory role in T cell activation and function. More recent studies have demonstrated that aged T cells produce more SAH than younger T cells post activation and that these same cultures produce SAH hydrolyase that cleaves of an adenosine group resulting in the activation of the A2aR and a suppression of cytokine expression and an increase in IL-10 production. These data suggest that alterations in T cell metabolism with age may influence homocysteine levels which may modulate cytokine expression and T cell phenotypes. Additional work has been initiated using mass spectroscopy to identify and quantitate various forms of Hcy in culture supernatants and serum/plasma. We believe that developing our ability to screen these Hcy mediators may elucidate potential mechanisms of immune activation associated with cardiovascular and inflammatory disease states. We are also currently completing several manuscripts for possible publication. Overall, Hcy appears to exert a number of differential effects on immune cells, which may alter immune function in the circulation and tissue microenvironment with age and inflammatory and/or autoimmune disease pathology. A greater understanding of the potential modulatory effects of Hcy and its metabolites on immune function may result in the development of potential therapeutic strategies to control and optimize immune responses with age, AIDS and in various age-associated disease states. This project will be terminated during the next fiscal year (FY2014).